Бесплатный автореферат и диссертация по биологии на тему

Характеристика спектра генов, экспрессия которых повышается при образовании В-клеточных не-ходжкинских лимфом, ассоциированных с ВИЧ-1

ВАК РФ 03.00.03, Молекулярная биология

Содержание диссертации, кандидата биологических наук, Калмырзаев, Болот Боромбаевич

СПИСОК СОКРАЩЕНИЙ.

ВВЕДЕНИЕ.

ЧАСТЬ I. ОБЗОР ЛИТЕРАТУРЫ.

Глава 1. Общая характеристика онкогенеза.

1.1. Онкогены.

1.2. Гены-супрессоры опухолей.

Глава 2. Вирусная трансформация клеток.

2.1. РНК-содержащие вирусы.

2.1.1. Вирусные онкогены как факторы онкогенеза.

2.1.2. Инсерционный мутагенез.

2.1.3. Вирусный онкогенез через трансактивацию клеточных генов.

2.2. ДНК-содержащие вирусы.

Глава 3. Молекулярные механизмы лимфомогенеза.

3.1. Генетические изменения, сопровождающие лимфомогенез.

3.1.1. Активация онкогенов в результате точковых мутаций.

3.1.2. Активация онкогенов в результате хромосомных перестроек.

3.1.3. Активация онкогена в результате амплификации гена.

3.2. Вирусы, ассоциированные с лимфомогенезом.

3.2.1. ДНК-содержащие вирусы приматов.

3.2.2. РНК-содержащие вирусы приматов.

Глава 4. ВИЧ-1 -ассоциированные лимфомы.

4.1. Клеточная классификация.

4.2. Молекулярные особенности ВИЧ-ассоциированных NHL.

4.3. Механизмы ВИЧ-ассоциированного лимфомогенеза.

4.3.1. Роль ВИЧ в лимфомогенезе.

4.3.3. Роль EBV в лимфомогенезе.

4.3.2. Дерегуляция онкогена с-тус.

4.3.4. Другие генетические аномалии, встречающиеся в ВИЧ-ассоциированных лимфомах.

4.3.5. Мультифакториальная модель.

4.4. SIV-ассоциированный лимфомогенез у обезьян.

ЧАСТЬ II. МАТЕРИАЛЫ И МЕТОДЫ.

ЧАСТЬ III. РЕЗУЛЬТАТЫ ЭКСПЕРИМЕНТОВ.

Глава 1. Анализ экспрессии генов, ассоциированных с В-клеточным лимфомогенезом, в клетках центробластной и иммунобластной лимфом.

1.1. Анализ экспрессии генов, ассоциированных с В-клеточным лимфомогенезом, методом RT-PCR.

1.2. Анализ экспрессии генов, ассоциированных с В-клеточным лимфомогенезом, методом гибридизации в точках.

Глава 2. Создание библиотек вычтенных лимфомоспецифических кДНК.

2.1. Вычитающая гибридизация.

2.2. Дифференциальный скрининг вычтенных библиотек кДНК.

Глава 3. Характеристика кДНК, селектированных из центробластной лимфомы

3.1. Последовательности, не имеющие гомологии с известными генами.

3.2. Последовательности, имеющие гомологию с клонированными ранее кДНК неизвестной природы (EST).

3.3. Последовательности, имеющие гомологию с известными генами.

3.4. Анализ слитой последовательности Xlg-set.

3.4.1. Характеристика слитого транскрипта AJg-set.

3.4.2. Геномная перестройка в цетробластной лимфоме ответственна за формирование химерного гена A,lg-set.

3.4.3. Дополнительные доказательства существования геномной перестройки AJg-set в клетках центробластной лимфомы.

Глава 4. Характеристика кДНК, селектированных из иммунобластной лимфомы . 70 4.1. Анализ экспрессии в клетках центробластной лимфомы митохондриальных генов, повышенно экспрессирующихся в клетках иммунобластной лимфомы,

ЧАСТЬ IV. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ.

1. Повышенная экспрессия гена set в клетках лимфом.

2. Хромосомная перестройка A,lg-set в клетках центробластной лимфомы.

3. Экспрессия митохондриальных генов, кодирующих белки окислительного фосфорилирования, в клетках лимфом.

ВЫВОДЫ.

Введение Диссертация по биологии, на тему "Характеристика спектра генов, экспрессия которых повышается при образовании В-клеточных не-ходжкинских лимфом, ассоциированных с ВИЧ-1"

Актуальность проблемы

Синдром приобретенного иммунодефицита (СПИД, англ. AIDS) был впервые описан в 1981 году. Очень скоро, в 1983 году, несколькими исследовательскими группами независимо был выделен и описан ретровирус, вызывающий этот синдром и названный вирусом иммунодефицита человека (ВИЧ, англ. HIV) (Gallo, Montagnier, 1987; Mitsuya et al., 1987; Haseltine et al., 1988). Первоначально в число злокачественных патологий, сопровождающих этот синдром, включали саркому Капоши (KS). Начиная с1984 года в ряде исследований, проводившихся независимо в нескольких исследовательских центрах, была показана взаимосвязь между злокачественными лимфоаденопатиями и СПИДом, и описан клинический спектр не-Ходжкинских лимфом (NHL), встречающихся у представителей этой группы больных (Ziegler et al., 1984). В результате в 1985 году Национальный Центр по контролю заболеваемости (CDC - Centers for Disease Control) (США) пересмотрел определение СПИДа, впервые включив туда агрессивную B-NHL, ассоциированную с ВИЧ (AIDSNHL) (Centers for Disease Control, 1985; Harnly et al., 1988; Kristal etal., 1988).

В настоящее время NHL занимают второе место, после саркомы Капоши, по частоте среди злокачественных новообразований, ассоциированных со СПИДом. В 1991 году в отчете CDC сообщалось, что ВИЧ-ассоциированные лимфомы составили 3-4% от всех заболеваний, определяемых при СПИДе. Заболеваемость NHL увеличивается почти параллельно с распространением эпидемии СПИДа и составляет 2-3% вновь диагностированных случаев СПИДа (Rabkin et al., 1994). Ряд авторов отмечали относительно постоянный уровень риска развития NHL в 1,6%-2% в год среди популяции, страдающей СПИДом (Moore et al., 1991; Gail et al., 1991; Pluda et al., 1990, 1993). Примерно у 57% пациентов диагноз СПИД предшествует началу NHL, но у 30% диагноз NHL ставится одновременно с ВИЧ-позитивностью и диагнозом СПИД (Knowles et al., 1988). NHL возникают, в конечном итоге, у 5-13% всех пациентов, страдающих СПИДом, либо на начальных, либо на поздних этапах развития СПИДа (Hamilton-Dutoit et al., 1991; Katz et al., 1994; Chan et al., 1995; Herndier et al., 1994). По некоторым оценкам ВИЧ-ассоциированные NHL составляют от 8% до 27% всех вновь диагностированных случаев лимфомы (Gail et al., 1991). Наблюдения, проведенные среди представителей групп риска (лица, страдающие гемофилией, наркоманы, гомосексуалисты), показали, что вероятность возникновения этой патологии у ВИЧ-инфицированных лиц в 12-38 раз выше, чем у 4

ВИЧ-негативных лиц того же возраста (Hessol et al., 1992; Koblin et al., 1998; Rabkin et al., 1992). По последним данным риск возникновения NHL у пациентов со СПИДом в некоторых странах в 200 раз выше, чем в целом в популяции людей (Doll, 1999).

Таким образом, NHL на сегодня представляют собой значительную клиническую проблему в спектре ВИЧ-ассоциированных заболеваний. Важность этой проблемы приобретает все большее значение в связи с нарастающими темпами распространения ВИЧ-инфекции в мире. Однако до сих пор не существует единой и ясной картины в понимании патогенетических и молекулярных основ развития злокачественных лимфом человека и, в частности, ВИЧ-ассоциированных лимфом. Тем более что ВИЧ-ассоциированные NHL по клинической картине, патоморфологии и молекулярным изменениям в клетках имеют ряд отличительных особенностей, которые позволяют выделить этот тип лимфом в отдельную группу. По этой причине для исследователей представляет большой интерес изучение молекулярно-генетических механизмов возникновения и развития этой группы лимфом у человека.

Одним из подходов к решению данной проблемы является определение генов, которые ведут себя активно в опухолевых клетках по сравнению с нормальными и способствуют процессу лимфомогенеза. Для решения подобной задачи, а также для последующего выяснения возможной роли идентифицированных генов в процессе злокачественного перерождения в последние годы стали широко использовать такие новые подходы как трансгеноз в сочетании с таргетингом, вычитающую гибридизацию, дифференциальный дисплей, сравнительную геномную гибридизацию и др. Использование этих современных методов позволяет существенно расширить наши представления о генетических основах онкогенеза. В частности, совершенствование таких методов как вычитающая гибридизация и дифференциальный дисплей привело к тому, что была получена новая информация о генах, экспрессия которых в раковых клетках значительно повышается или понижается (Schraml et al., 1994; Paciucci et al. 1996; Wu et al., 1996, 1997; Sarto et al., 1997). Анализ таких генов в злокачественных клетках, позволил обнаружить новые онкогены и гены-супрессоры, показать участие в онкогенезе известных и ранее неизвестных генов. 5

Цели и задачи исследования

Основная цель настоящей работы - изучение молекулярных механизмов В-клеточного лимфомогенеза, ассоциированного с ВИЧ-инфекцией.

В соответствии с поставленной целью были сформулированы следующие задачи:

1. С помощью вычитающей гибридизации определить спектр генов, повышающих свою транскрипцию в клетках ВИЧ-ассоциированных В-клеточных не-Ходжкинских лимфом человека.

2. Провести сравнительный анализ наборов генов, харктеризующихся повышенной транскрипцией в двух типах ВИЧ-ассоциированных лимфом.

Результаты получены как лично автором, так и совместно с сотрудниками и аспирантами Лаборатории молекулярной генетики ретровирусов и СПИДа Отдела клеточной и вирусной молекулярной генетики Института молекулярной генетики РАН. 6

ЧАСТЬ I. ОБЗОР ЛИТЕРАТУРЫ

Заключение Диссертация по теме "Молекулярная биология", Калмырзаев, Болот Боромбаевич

ВЫВОДЫ

1. Определён спектр генов, характеризующихся повышенным уровнем транскрипции в клетках центробластной В-клеточной не-Ходжкинской лимфомы человека, ассоциированной с ВИЧ-1-инфекцией. Повышенная экспрессия характерна для онкогена set, гена константной области легкой цепи иммуноглобулина Я1д, интерферон-индуцируемого гена 6-16, митохондриальных генов, кодирующих белки окислительного фосфорилирования НАДН-дегидрогеназу 1 и НАДН-дегидрогеназу 4, гена митохондриальной 16S рРНК и ряда ранее неизвестных генов.

2. Определён спектр генов, характеризующихся повышенным уровнем транскрипции в клетках иммунобластной В-клеточной не-Ходжкинской лимфомы человека, ассоциированной с ВИЧ-1-инфекцией. Повышенная экспрессия характерна для митохондриальных генов, кодирующих белки окислительного фосфорилирования НАДН-дегидрогеназу 4, АТФ-синтазу 6, цитохром с оксидазу 2, цитохром Ь, гена митохондриальной 16S рРНК.

3. В клетках центробластной лимфомы обнаружена и охарактеризована, уникальная хромосомная перестройка с участием гена константной области легкой цепи иммуноглобулина Я1д и онкогена set, приводящая, по-видимому, к активации транскрипции этих двух генов.

4. Для обоих типов ВИЧ-ассоциированных лимфом человека характерна повышенная экспрессия митохондриальных генов НАДН-дегидрогеназы 4, цитохром с оксидазы 2 и 16S рРНК. Это позволяет предположить, что повышение экспрессии генов, кодирующих белки окислительного фосфорилирования, ассоциировано с процессом лимфомогенеза.

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